Mutations in the EGFR kinase domain are a major cause of non-small cell lung cancer, and treatment with an EGFR-directed tyrosine kinase inhibitor (TKI) is the standard of care for patients with advanced EGFR-mutant lung cancer. However, resistance limits the long-term efficacy of these drugs. Understanding and overcoming acquired resistance to EGFR TKIs and to other targeted therapies is a central problem in cancer medicine. In analogy with the ability of triple-cocktail therapy to overcome the mutagenic repertoire of the HIV virus, we reason that simultaneous treatment with multiple agents targeting mutant EGFR may prevent emergence of resistance mutations, leading to more durable responses. However, at present suitable compounds with alternative mechanisms of action are not available. Like the vast majority of TKIs, all current EGFR TKIs target the ATP-site of the kinase. As described in our preliminary results, we are developing novel allosteric EGFR inhibitors that target a distinct pocket in the kinase. Enzyme kinetic studies and a co-crystal structure confirm their allosteric mechanism of action. These agents potently inhibit the EGFR T790M mutant, but are inactive on wild type EGFR. The distinct mechanism of action and binding site of these allosteric inhibitors, together with their lack of activity on WT EGFR and other protein kinases makes them especially attractive as candidates for combination therapy. We are a highly collaborative, multidisciplinary research team with deep interest in EGFR-mutant lung cancer and a record of successful inhibitor discovery. Our expertise spans kinase structure and mechanism (Eck, Co-PI), medicinal chemistry (Gray, Co-PI), cellular pharmacology (Jnne) and mouse models (Wong). Our goal is to develop first-in-class mutant-selective, allosteric EGFR inhibitors with single- agent activity in cellular and mouse models of EGFR-mutant lung cancer. In order to accomplish this goal, we further probe the binding repertoire of the allosteric pocket with virtual ligand discovery coupled with biochemical and cellular inhibition assays (Aim 1). We apply these tools and findings to drive optimization of these allosteric inhibitors through a focused medicinal chemistry effort (Aim 2), and we establish the efficacy of our optimized compounds in preclinical studies using genetically engineered mouse models of EGFR mutant lung cancer (Aim 3). Successful completion of these aims will provide proof of concept for a new class of targeted therapeutics for EGFR-mutant lung cancers that are resistant to all current agents.